Compounds and methods for treatment of sickle cell disease or complications associated therewith

ABSTRACT

Compounds, compositions and methods are provided for treatment of sickle cell disease or a complication associated therewith, or graft versus host disease, in an individual. More specifically, the use of particular glycomimetics for the treatment is described.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 60/959,984 filed Jul. 18, 2007; whichapplication is incorporated herein by reference in its entirety.

BACKGROUND

1. Technical Field

The present invention relates generally to compounds, compositions andmethods for treating sickle cell disease or complications associatedtherewith, and more specifically to the use of particular glycomimeticsfor the treatment. The glycomimetics may also be used to treat graftversus host disease.

2. Description of the Related Art

Sickle cell disease is an inheritable hematological disorder based on amutation in the β-globin gene of hemoglobin. Upon deoxygenation, thismutated hemoglobin polymerizes and causes a shape change (sickling) ofthe red blood cell. This change in red blood cells leads to obstructionof blood vessels causing a wide variety of complications such as stroke,pulmonary hypertension, end-organ disease and death.

In addition to the fatal or potentially fatal complications, there areserious non-fatal complications of sickle cell disease such as pain. Theseverity of the pain may vary, but normally requires some form ofmedical attention. Hospitalization may be necessary.

In the U.S. alone, approximately 70,000-80,000 people suffer from sicklecell disease. Sickle cell disease is estimated to affect one of every1,300 infants in the general population, and one of every 400 of Africandescent. Currently, there is no cure for sickle cell disease. Thedisease is chronic and lifelong. Life expectancy is typically shortened.

Accordingly, there is a need in the art for the treatment of sickle celldisease or the complications associated therewith. The present inventionfulfills these needs and further provides other related advantages.

BRIEF SUMMARY

Briefly stated, compounds, compositions and methods for treating sicklecell disease or the complications associated therewith, or graft versushost disease, are provided. In the present invention, the compounds usedfor treatment comprise, or consist of, a particular glycomimetic. Such acompound may be combined with a pharmaceutically acceptable carrier ordiluent to form a pharmaceutical composition.

In one embodiment, the present invention provides a method for thetreatment of sickle cell disease or a complication associated therewithin an individual who is in need thereof, comprising administering to theindividual a compound in an amount effective for treatment, the compoundwith the formula:

wherein

L=linker group; and

n=0-1.

In one embodiment, the present invention provides a method for thetreatment of graft versus host disease in an individual who is in needthereof, comprising administering to the individual a compound in anamount effective for treatment, the compound with the formula:

wherein

L=linker group; and

n=0-1.

In other embodiments, the above compounds or compositions thereof may beused in the manufacture of a medicament, for any of the uses recitedherein.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a diagram illustrating the synthesis of a component ofCompound #1.

FIG. 2 is a diagram illustrating the synthesis of a component ofCompound #1.

FIG. 3 is a diagram illustrating the modification of the component ofFIG. 1.

FIG. 4 is a diagram illustrating the reaction of the components of FIGS.2 and 3 to form Compound #1. Compound XIX of FIG. 2 is reacted withethylenediamine (EDA) to form EDA-XIX.

FIG. 5 is a diagram illustrating the synthesis of Compound #2. CompoundXIX of FIG. 2 is reacted with ethylenediamine (EDA) to form EDA-XIX.

FIG. 6 is a schematic of the protocol for inducing a vaso-occlusivecrisis (VOC) in sickle cell-affected mice and recording blood flow.

FIG. 7 shows the effects of a compound or anti-E&P monoclonal antibodieson blood flow in the sickle cell mouse.

FIG. 8 shows the effects of a compound or anti-E&P monoclonal antibodieson the adhesion of sickle red blood cells to leukocytes in sickle cellmice in which a vaso-occlusive crisis was induced.

FIG. 9 is a schematic of a sickle cell mouse model for use with bothprevention and treatment protocols. The black arrows underneath Comp. #2are for the prevention protocol. The gray arrow underneath Comp. #2 isfor the treatment protocol. Recording is started at the 90 minute markfor the prevention protocol. Recording is started at the 120 minute markfor the treatment protocol.

FIG. 10 shows the effects of a compound on delayed treatment of VOC insickle cell mice as measured by blood flow rate. Compound #2 normalizesthe rate of blood flow. The control is phosphate buffered saline (PBS)without compound.

FIG. 11 shows the effects of a compound on delayed treatment of VOC insickle cell mice as measured by adherent white blood cells (WBCs).Compound #2 causes a significant reduction in the number of WBCsadherent to the vascular endothelium. The control is PBS withoutcompound.

FIG. 12 shows the effects of a compound on delayed treatment of VOC insickle cell mice as measured by interactions of sickle red blood cells(RBCs) with leukocytes (white blood cells). Compound #2 dramaticallyreduces interactions of sickle RBCs with leukocytes. The control is PBSwithout compound.

FIG. 13 depicts Kaplan-Meier survival curves. The curve with blackdiamond symbols is for Compound #2. The curve with gray squares is forthe control of PBS without compound.

DETAILED DESCRIPTION

As noted above, the present invention provides compounds, compositionsand methods for the treatment of sickle cell disease or a complicationassociated therewith, or graft versus host disease, in an individual.

Compounds useful in the compositions and methods of the presentinvention include embodiments with the formula:

In the above formula, “L” represents a linker. There may be no linkerspresent (i.e., “n” is 0) or a linker may be present (i.e., “n” is 1).Where no linker is present, the compound is with the formula:

Where n is 1, a linker is present. A linker may include a spacer group,such as —(CH₂)_(p)— or —O(CH₂)_(p)— where p is generally about 1-20(including any whole integer range therein). Other examples of spacergroups include a carbonyl or carbonyl containing group such as an amide.

Embodiments of linkers include the following:

Other linkers, e.g., polyethylene glycols (PEG) or—C(═O)—NH—(CH₂)_(p)—C(═O)—NH₂ where p is as defined above, will befamiliar to those in the art or in possession of the present disclosure.

In another embodiment, the linker is

which produces:

In another embodiment, the linker is

which produces:

All compounds of the present invention or useful thereto (e.g., forpharmaceutical compositions or methods of treating), includephysiologically acceptable salts thereof. Examples of such salts are Na,K, Li, Mg, Ca and Cl.

Compounds as described herein may be present within a pharmaceuticalcomposition. A pharmaceutical composition comprises one or morecompounds in combination with (i.e., not covalently bonded to) one ormore pharmaceutically or physiologically acceptable carriers, diluentsor excipients. Such compositions may comprise buffers (e.g., neutralbuffered saline or phosphate buffered saline), carbohydrates (e.g.,glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptidesor amino acids such as glycine, antioxidants, chelating agents such asEDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/orpreservatives. Within yet other embodiments, compositions of the presentinvention may be formulated as a lyophilizate. Compositions of thepresent invention may be formulated for any appropriate manner ofadministration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous, orintramuscular administration.

The compositions described herein may be administered as part of asustained release formulation (i.e., a formulation such as a capsule orsponge that effects a slow release of compound followingadministration). Such formulations may generally be prepared using wellknown technology and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Carriers for use within such formulations are biocompatible, andmay also be biodegradable; preferably the formulation provides arelatively constant level of compound release. The amount of compoundcontained within a sustained release formulation depends upon the siteof implantation, the rate and expected duration of release and thenature of the condition to be treated or prevented.

The above described compounds including equivalents thereof are usefulin methods of the present invention as it relates to sickle cell diseaseand as it relates to graft versus host disease. In an embodiment, anindividual who is in need of treatment for sickle cell disease or acomplication associated therewith is administered at least one (i.e.,one or more) of the above described compounds in an amount effective forthe treatment. As used herein, the term “treatment” (includingvariations such as “treating”) includes prevention. For example, acomplication associated with sickle cell disease may not have presenteditself in an individual with the disease, and a compound may beadministered to prevent presentation of the complication in theindividual. Sickle cell disease and complications associated therewithinclude, for example, anemia, red blood cells becoming stuck in bloodvessels, ischemia, infarction, stroke, acute chest crisis, splenicsequestration crisis, shortened life expectancy, organ damage andperiodic or chronic pain.

In another embodiment, an individual who is in need of treatment forgraft versus host disease (GVHD) is administered at least one (i.e., oneor more) of the above-described compounds in an amount effective for thetreatment. GVHD commonly arises in patients post stem celltransplantation. A preferred route of administration is via an orallyavailable formulation.

The term “treatment,” as set forth above, refers to any of a variety ofpositive effects from the treatment including, for example, eradicatinga complication associated with the disease, relieving to some extent acomplication, slowing or stopping progression of the disease, andprolonging the survival time of the recipient. The treatment may be usedin conjunction with one or more other therapies for sickle cell diseaseor complications associated therewith, or therapies for graft versushost disease.

The above described compounds may be administered in a mannerappropriate to the disease to be treated. Appropriate dosages and asuitable duration and frequency of administration may be determined bysuch factors as the condition of the patient, the type and severity ofthe patient's disease and the method of administration. In general, anappropriate dosage and treatment regimen provides the compound(s) in anamount sufficient to provide therapeutic and/or prophylactic benefit.Within particularly preferred embodiments of the invention, a compoundmay be administered at a dosage ranging from 0.001 to 1000 mg/kg bodyweight (more typically 0.01 to 1000 mg/kg), on a regimen of single ormultiple daily doses. Appropriate dosages may generally be determinedusing experimental models and/or clinical trials. In general, the use ofthe minimum dosage that is sufficient to provide effective therapy ispreferred. Patients may generally be monitored for therapeuticeffectiveness using assays suitable for the condition being treated,which will be familiar to those of ordinary skill in the art.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Synthesis of BASA (FIG. 1)

Synthesis of compound 4: 3-nitro-benzyl iodide (1) (48.3 g) is added toan aqueous solution (pH 11) of commercially available (Aldrich ChemicalCo., Milwaukee, Wis.), 8-aminonaphthalene-1,3,5-trisulfonic acid (2)(29.5 g) with stirring at room temperature. pH of the solution isadjusted to 1 and after evaporation of the solvent, the product 3 (6.4g) is precipitated out from ethanol.

Platinum catalyzed hydrogenation of compound 3 affords compound 4 (theBASA of FIG. 1) in 96% yield.

Example 2 Synthesis of Glycomimetic (FIG. 2)

Synthesis of intermediate II: (−)-Shikimic acid (20 g) in MeOH (200 ml)and sulfuric acid (2 ml, 98%) are stirred at room temperature (rt) for50 h. The reaction mixture is neutralized with 2N aqueous NaOH in thecold. After evaporation to dryness, the residue is purified by silicagel chromatography to afford II (19.2 g).

Synthesis of intermediate III: Methyl shikimate (II, 10 g), 2,2dimethoxypropane (10 ml) and p-TsOH (0.8 g) are dissolved inacetonitrile (125 ml) and stirred at rt for 1 h. The reaction mixture isthen neutralized with triethylamine (2 ml) and evaporated to dryness.The residue is chromatographed on silica gel to yield III (11 g).

Synthesis of intermediate IV: The shikimic acid derivative III (10 g)and PtO₂/C (10%, 250 mg) in MeOH (40 ml) are hydrogenated at rt undervigorous stirring. After 16 h the reaction mixture is filtered overcelite and evaporated to dryness. The residue is chromatographed onsilica gel to yield IV.

Synthesis of intermediate V: To a solution of IV (8 g) in DCM (100 ml)at 0° C. are added pyridine (12 ml), acetic anhydride (7 ml) and a DMAP(25 mg). The reaction mixture is stirred at rt for 1 h, and diluted withEtOAc (250 ml). After washing with 0.5 M aqueous HCl (3×50 ml),saturated solution of KHCO₃ (3×50 ml) and brine (3×50 ml), the combinedorganic layers are dried (Na₂SO₄) and evaporated to dryness. The residueis purified by chromatography on silica gel to yield V (6.8 g).

Synthesis of intermediate VI: A solution of V (6.0 g) in acetic acid (30ml, 80%) is stirred at 80° C. for 1 h. Solvent is evaporated off and theresidue is purified by chromatography on silica gel (DCM/MeOH 14:1) toyield VI (3.6 g).

Synthesis of intermediate VII: A solution of VI (3 g) and p-TsCl (3.5 g)in pyridine (30 ml) is stirred at rt for 6 h. MeOH (5 ml) is added andthe solvent is evaporated at reduced pressure, the residue dissolved inEtOAc (3×150 ml) and the organic layers are washed with 0.5 M aqueousHCl (0° C.), water (cold) and brine (cold). The combined organic layersare dried (Na₂SO₄), filtered on Celite and evaporated to dryness. Theresidue is purified by chromatography on silica gel (toluene/EtOAc 4:1)to yield VII (3.7 g).

Synthesis of compound VIII: A solution of VII (3 g) and NaN₃ (2.5 g) inDMF (20 ml) is stirred at 80° C. The reaction mixture is cooled to rtand diluted with EtOAc (200 ml) and water (50 ml). The organic layer isadditionally washed twice with water (2×50 ml) and once with brine (50ml). All aqueous layers are extracted twice with EtOAc (2×50 ml). Thecombined organic layers are dried with Na₂SO₄, filtered and the solventis evaporated off. The residue is purified by chromatography on silicagel (petroleum ether/EtOAc 5:2) to give VIII (2.2 g).

Synthesis of compound X: To a solution of ethyl2,3,4-tri-O-benzyl-α-L-fucothiopyanoside IX (1.5 g) in DCM (3 ml),bromine (150 μl) is added at 0° C. under argon. After 5 min the coolingbath is removed and the reaction mixture is stirred for additional 25min at rt. Cyclohexene (200 μl) is added and the reaction mixture isadded to a solution of VIII (400 mg), (Et)₄NBr (750 mg) and powdered 4 Åmolecular sieves in DCM (10 ml) and DMF (5 ml). After 16 h,triethylamine (1.5 ml) is added and stirred for an additional for 10min, diluted with EtOAc (50 ml) and washed with sat. aqueous NaHCO₃,water and brine. The aqueous layers are extracted twice with EtOAc (2×50ml). The combined organic layers are dried (Na₂SO₄), filtered andevaporated to dryness. The residue is purified by chromatography onsilica gel (toluene/EtOAc 9:1) to yield X (700 mg).

Synthesis of compound XI: To a solution of X (1.5 g) in MeOH (20 ml) isadded freshly prepared NaOMe (80 mg) and the reaction mixture is stirredin a pressure tube at 80° C. for 20 h. The reaction mixture is cooled tort and neutralized with acetic acid. Solvent is evaporated to drynessand the residue is dissolved in ether. Freshly prepared diazomethane isadded and the excess diazomethane is neutralized with acetic acid.Solvent is evaporated off to give XI (1.25 g).

Synthesis of building block XV: This synthesis is done exactly in sameway as described previously (Helvetica Chemica Acta 83:2893-2907(2000)).

Synthesis of compound XVI: A mixture of XI (1.6 g), XV (3 g) andactivated powdered molecular sieves 4 Å (1 g) in DCM (17 ml) is stirredat rt under argon for 2 h. Then DMTST (2 g) is added in 4 equal portionsover a period of 1.5 h. After 24 h the reaction mixture is filtered overCelite and the filtrate is diluted with DCM (100 ml). The organic layeris washed with sat. aqueous NaHCO₃ and brine and the aqueous layers areextracted twice with DCM. The combined organic layers are dried(Na₂SO₄), filtered and evaporated to dryness. The residue is purified bychromatography on silica gel (toluene/EtOAc 8:1) to yield XVI (1.5 g).

Synthesis of compound XVII: To a solution of XVI (500 mg) and oroticacid chloride (500 mg) in dichloromethane (10 ml) is added a solution oftriphenylphosphine (500 mg in 5 ml dichloromethane) dropwise during 10min. The reaction mixture is stirred at rt for 25 h and the solvent isevaporated off. The residue is purified (chromatography on silica gelDCM/MeOH 19:1) to give XVII (250 mg).

Synthesis of compound XVIII: To a solution of XVII (200 mg) indioxane-water (5:1, 12 ml) is added 10% Pd—C (100 mg) and the reactionmixture is stirred vigorously under hydrogen (55 psi) for 24 h. Catalystis filtered through a bed of celite and the solvent is evaporated off.Residue is purified by silica gel chromatography to give compound XVIII(150 mg).

Synthesis of XIX: To a solution of compound XVIII (145 mg) in MeOH (5ml) is added a solution of NaOMe in MeOH (25%, 0.025 ml) and thereaction mixture is stirred at rt for 4 h, neutralized with acetic acidand the solvent is evaporated off. Residue is dissolved in water andpassed through a bed of Dowex 50wX-8 (Na-form) resin. Water wash isevaporated off to afford compound XIX (100 mg).

Synthesis of EDA-XIX: XIX (80 mg) is heated at 70° C. withethylenediamine (EDA) (1 ml) with stirring for 5 h. Solvent isevaporated off and the purified by sephadex G-25 column to give EDA-XIX(82 mg).

Example 3 Synthesis of PEGylated BASA (FIG. 3)

To a solution of 3,6-dioxaoctanedioic acid (PEG, 200 mg, AldrichChemical Co., Milwaukee, Wis.) in DMF (1 ml) is added Hunig base (0.4ml), and then HATU (0.35 g) is added after 5 min. The solution isstirred at RT for 10 min. and then a solution of the BASA of Example 2(50 mg) in DMF (0.1 ml) is added. The reaction mixture is stirred for 4h at rt and the solvent is evaporated off. The residue is purified byhplc (reverse-phase C18 column) to give XX (40 mg).

Example 4 Synthesis of Glycomimetic-BASA Compound #1 (FIG. 4)

To a solution of XX from Example 3 (0.015 g) in DMF (0.1 ml), is addedHunig base (0.015 ml) and then HATU (0.007 g). The reaction mixture isstirred for 10 min at RT. A solution of EDA-XIX from Example 2 (0.010 gin DMF ml) is added and the reaction mixture is stirred at RT for 8 h.Solvent is evaporated off and the residue is purified by sephadex G-25chromatography to give Glycomimetic-BASA #1 of FIG. 4 (0.008 g).

Example 5 Synthesis of Glycomimetic-BASA Compound #2 (FIG. 5)

Synthesis of compound XXI: To a solution of 3,6-dioxaoctanedioic acid(PEG, 200 mg, available commercially) in DMF (1 ml) is added Hunig base(0.4 ml) and then HATU (0.35 g) is added after 5 min. The solution isstirred at RT for 10 min and then solution of8-aminonaphthalene-1,3,6-trisulfonic acid (50 mg, availablecommercially) in DMF is added. The reaction mixture is stirred for 4 hat RT and the solvent is evaporated off. The residue is purified by hplc(reverse-phase C18 column) to give XXI (25 mg).

Synthesis of compound XXII: This synthesis is performed in the same wayas described in example 4 except using EDA-XIX from example 2 and XXI togive compound XXII (4 mg).

Example 6 Effects of Compound # 2 on Microvascular Flow in Sickle CellMice as Determined by Intravital Microscopy

Sickle cell disease is a genetic condition caused by a point mutation(β^(s)) in the β-chain of hemoglobin. This single mutation leads toabnormal microvascular flow, endothelial activation and episodicvaso-occlusion. Impairment of blood flow is responsible for the severepain, end organ damage and eventual death of these patients.

An animal model of sickle cell disease exists in fully chimeric miceconstructed by bone marrow transplantation and expressing >97% humanglobin containing the β^(s) mutation. A vaso-occlusive crisis is inducedby stimulation with TNFα and is monitored by observing and recordingblood flow by intravital microscopy of the venules in the cremastermuscle. A schematic diagram of the protocol is shown in FIG. 6.

Each mouse is prepared by cannulating the right carotid artery and byundergoing a tracheotomy to facilitate ventilation under anesthesia.Twenty (20) minutes prior to stimulation with TNFα, the cremaster muscleis gently exteriorized and the venules are set on a microscope stage forobservation and recording of blood flow. Just prior to administration ofTNFα and again prior to the recording of the venules (90 min later),Compound # 2 is administered through the cannulated carotid artery anddata is obtained by monitoring blood flow and recording time of death.

The effects of Compound # 2 on blood flow in the sickle cell mouse areshown in FIG. 7. In the control mice, in which only vehicle is injected,blood flow is very slow and indicative of the sickle disease state afterstimulation with TNFα. Either Compound # 2 or a mixture of antibodiesagainst E- and P-selectin have dramatic effects by restoring blood flowto a velocity observed in normal mice.

During a vaso-occlusive crisis which is simulated in the sickle cellmouse model, blood cells adhere to each other, form aggregates, anddecrease blood flow to vital organs, resulting in organ failure, and insevere cases, death. The effects of Compound # 2 on the adhesion ofsickle red blood cells (RBC) to leukocytes was determined in sickle cellmice in which TNFα was used to induce a vaso-occlusive crisis. As shownin FIG. 8, both Compound # 2 and a mixture of antibodies against E- andP-selectins provide significant inhibition of adhesion among these celltypes.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

1. A method for the treatment of sickle cell disease or a complicationassociated therewith in an individual who is in need thereof, comprisingadministering to the individual a compound in an amount effective fortreatment, the compound with the formula:

wherein L=linker group; and n=0-1.
 2. The method according to claim 1wherein in the compound n=0.
 3. The method according to claim 1 whereinin the compound n=1 and L is

where the N of L is attached to terminal C of C(═O) of the compound. 4.The method according to claim 1 wherein in the compound n=1 and L is

where the N of L is attached to terminal C of C(═O) of the compound. 5.The method of any one of claims 1-4 wherein the compound is incombination with a pharmaceutically acceptable carrier or diluent.